DE102021134613A1 - Method for concentrating at least one biological target substance in a sample liquid and method for automated online detection of at least one biological target substance in a sample liquid - Google Patents

Abstract

The invention relates to a method for concentrating at least one biological target substance in a sample liquid, in particular one that can be carried out automatically by means of control electronics, comprising: - Dosing and transporting a first initial volume of the sample liquid via a liquid supply line (25) into a first concentrator chamber (8) of a concentrator unit (3, 33); and- generating concentrated sample liquid by reducing the first initial volume of the sample liquid in the concentrator unit (3, 33) and thereby concentrating the at least one biological target substance contained in the first volume of the sample liquid using a first quantity of a superabsorbent (24), wherein the reduction and the associated concentration are carried out by incubating a mixture formed from the first initial volume of the sample liquid with the first quantity of the super absorber (24). The invention also relates to a device for carrying out the method.

Description

The invention relates to a method for concentrating at least one biological target substance in a sample liquid, e.g. water or waste water, and a concentrator unit for the automated concentration of at least one biological target substance in a sample liquid. The invention also relates to a method for automated online detection of at least one biological target substance using molecular genetic methods in a sample liquid, e.g. water or waste water, and an online analysis device for detecting at least one biological target substance using molecular genetic methods in a sample liquid.

The detection of biological parameters plays an increasingly important role in the process industry, for example in the monitoring and cleaning of industrial water or sewage water, in food production and in the pharmaceutical and life sciences sector, for example in the monitoring of biotechnological processes carried out in fermentors. Target substances to be detected or monitored can be, for example, viruses, bacteria and plasmid-associated bacterial resistance genes.

Such biological target substances can be detected in liquids using molecular genetic methods, preferably using amplification techniques such as PCR or real-time PCR. For this purpose, samples of the liquid to be analyzed are taken and analyzed in specialized laboratories that have the necessary equipment systems. Manually manageable or automatic sampling devices and sample collectors are known for automated sampling from processes, e.g. The collected samples are usually transported to a laboratory for further analysis, where the extraction of the sample nucleic acids and the specific and possibly quantitative detection of the biological target substance is carried out manually or at least partially automatically.

For example, in the field of water and waste water analysis, online analysis devices are known which take a sample and carry out a quantitative determination of a mostly inorganic analyte in the sample in a fully automated manner. Such an online analysis can be carried out continuously or discontinuously.

Out of U.S. 2015/0224502 A1 a sample collection device for flow-through sampling in bodies of water is known. The device has several flow-through sample cartridges that are set up to receive water from the water to be monitored and, if necessary, to retain samples of components of the liquid or solids in a filter or adsorption medium, while the water received is returned from the cartridges to the body of water is returned. The samples can either be stored for later laboratory analysis or analyzed directly in the device. For this purpose, for example, biological substances adsorbed on the filter or adsorption medium can be released as lysate and made available to an analysis module for further analysis, eg by means of qPCR.
When the concentrations of the target substances in the liquid to be analyzed are low, the problem arises of providing a sufficient quantity of the target substance for the subsequent analysis when taking the sample. With a flow-through sampling like that in U.S. 2015/0224502 A1 is described, dissolved substances or solids present in the water, even in low concentrations, can be retained in the filter medium by passing through a correspondingly large volume of water. However, this is only practical if the liquid to be analyzed can be fed back into the medium without restrictions after the sample, as is the case, for example, with water analysis. When monitoring processes, for example in water treatment, the food industry or in the pharmaceutical sector, it is often not possible to reintroduce the removed liquid. There is a need here for a method for the efficient concentration of sample components to be detected, which makes it possible to keep the liquid volume to be removed from the process for the analysis low.

Another disadvantage of the in U.S. 2015/0224502 A1 The device described consists in that the substance retained in the filter or adsorption medium is passed into the analysis module by lysis and elution from the filter or adsorption medium. On the one hand, it is to be expected that only a relatively small proportion of the target substance from the filter or adsorption medium gets into the analysis module, and on the other hand, a certain minimum volume of liquid is required to flush out the filter, so that the solution entering the analysis module is relatively small has a large volume with a correspondingly low concentration of the target substance.

The object of the invention is to enable improved automated online detection of a biological target substance in a sample liquid and an improved method and an improved device for automated Specify online detection of a biological target substance in a sample liquid. In particular, the method and the device should enable an efficient concentration of the target substance and a low-loss forwarding of the sample liquid with the concentrated target substance to a subsequent detection unit.

This object is achieved by the method for concentrating at least one biological target substance in a sample liquid according to claim 1, by the method for automated online detection of at least one biological target substance in a sample liquid using an online analysis device according to claim 14, by the concentrator unit for automated implementation of a concentration of a target substance in a sample liquid according to claim 20 and by the online analysis device for detecting at least one biological target substance in a sample liquid according to claim 26. Advantageous configurations are specified in the dependent claims.

The solution is based on the use of so-called super absorbers, with which any aqueous sample liquid can be processed in order to concentrate the biomolecules contained in the sample liquid.

Superabsorbers (superabsorbent polymers, SAP) are plastics that are able to absorb many times their own weight in polar liquids. These are primarily water or aqueous solutions. When the liquid is absorbed, the superabsorbent swells and forms a hydrogel. Hydrogels can form any crosslinked polymer that is polar (eg, polyacrylamide, polyvinylpyrrolidone, amylopectin, gelatin, cellulose). In most cases, however, a copolymer of acrylic acid (propenoic acid, H2C=CH-COOH) or sodium acrylate (sodium salt of acrylic acid, H2C=CH-COONa) on the one hand and acrylamide on the other is used, although the ratio of the two monomers to one another can vary. In addition, a so-called core cross-linker (CXL) is added to the monomer solution, which connects the long-chain polymer molecules formed with one another in places by chemical bridges, also referred to as cross-linking. These bridges make the polymer insoluble in water. This so-called base polymer is possibly subjected to what is known as surface post-crosslinking, called surface cross-linking (SXL). Another chemical is applied to the surface of the particles, which, when heated, forms a second network only on the outer layer of the grain. This shell supports the swollen gel to hold it together even under external stress (movement, pressure).

For example, the product is used conventionally as white granules with particle sizes of 100 to 1000 µm. It is mainly used in baby diapers, sanitary napkins, in incontinence care, in bandages and in small quantities in cable sheathing for deep-sea pipelines. Other areas of application are so-called gel beds, gel-forming extinguishing agents in firefighting, as a mechanical stabilizer for cut flowers in a vase or as an additive for potting soil to permanently store water. Here, however, potassium hydroxide-neutralized acrylic acid is used because of its better environmental compatibility. In the form of spherical particles, the use of superabsorbents under designations such as "water beads", "aqua beads" or "water beads" is known as toys. These are super absorbers, which are commercially available in the form of spheres of variable size (submillimeters to centimeters).

The invention described here was based on the following unexpected observation. Commercially available so-called water beads (commercially available under the designations aqua beads, water beads, water beads, or gel beads, among others) were added to a liquid volume of 1 liter. These water beads are made from a super absorber material. The liquid was surface water that had been taken from a fire-fighting pond with suspended matter. After an incubation period, the beads swelled to many times their original volume. The volume of the liquid portion of the mixture of the liquid and the water beads was reduced. Surprisingly, it turned out that the suspended matter in the liquid was not absorbed by the swelling beads. The liquid portion of the mixture including the suspended solids (volume 400 ml) was transferred to a new vessel. A sample with a volume of 50 ml was taken from this liquid portion.

A comparison sample with a volume of 50 ml was taken directly from the liquid, i.e. the surface water mentioned, without previously concentrating the liquid according to the method described. Both samples were centrifuged at 5000 x g for 10 min. The supernatant was removed and the pellet used for nucleic acid extraction. The nucleic acid extraction was carried out using a commercial kit (innuprep Stool DNA Mini Kit; IST Innuscreen GmbH). The DNA from both samples was then examined for the amount of total bacterial count using real-time PCR.

This showed that fewer bacteria were detected in the untreated comparison sample than in the sample concentrated with the superabsorbent. This means that the bacteria contained in the liquid have not been absorbed into the water beads. They were concentrated and formed part of the remaining liquid portion of the mixture of the liquid and the superabsorbent beads. These observations could subsequently also be confirmed for other biomolecules (viruses or eukaryotic cells): After adding the superabsorber to a volume of liquid containing these biomolecules, the volume was reduced and the biomolecules were concentrated in the remaining liquid. It was even more surprising that the method described can also be used to concentrate proteins and free genomic DNA that are present in low concentrations in an aqueous solution.

The object on which the invention is based could be achieved on the basis of this observation.

• - Dosieren und Transportieren eines ersten initialen Volumens der Probeflüssigkeit über eine Flüssigkeitszuleitung in eine erste Konzentratorkammer einer Konzentrator-Einheit; und
• - Erzeugen von aufkonzentrierter Probeflüssigkeit durch Reduzieren des ersten initialen Volumens der Probeflüssigkeit in der Konzentrator-Einheit und damit verbundenem Aufkonzentrieren von in dem ersten Volumen der Probeflüssigkeit enthaltenen Biomolekülen mittels einer ersten Menge eines Superabsorbers, wobei das Reduzieren und das damit verbundene Aufkonzentrieren durch Inkubieren eines aus dem ersten initialen Volumen der Probeflüssigkeit mit der ersten Menge des Superabsorbers gebildeten Gemisches erfolgt.

The method according to the invention for concentrating at least one biological target substance in a sample liquid comprises:

• - Dosing and transporting a first initial volume of the sample liquid via a liquid supply line into a first concentrator chamber of a concentrator unit; and
• - Generating concentrated sample liquid by reducing the first initial volume of the sample liquid in the concentrator unit and the associated concentration of biomolecules contained in the first volume of the sample liquid using a first amount of a superabsorbent, the reduction and the associated concentration by incubating a the mixture formed by the first initial volume of the sample liquid with the first amount of the superabsorbent.

The volume-reduced sample liquid obtained after incubation with the superabsorber with a correspondingly increased concentration of the biomolecules contained therein or the at least one biological target substance contained therein is also referred to here and below as “concentrated sample liquid”.

By reducing the initial volume of the sample liquid to an adjustable volume after incubation with the superabsorbent material, which is accompanied by an increase in the concentration of the biological target substance in the remaining volume of the sample liquid, a method compared to the method described in the prior art can be used the biomolecules are released after binding to a filter medium by rinsing out the filter medium, a comparatively high proportion of the biomolecules obtained from the first initial volume of the sample liquid are concentrated in a small volume of liquid. Thus, the method according to the invention not only brings about an efficient concentration of the target substance to be determined in the sample liquid, but also makes it possible, even with a low concentration of the target substance in the sample liquid to be examined, to keep the volume determined for the subsequent analysis, e.g. from a process, comparatively small to choose. This makes the method universally suitable for monitoring liquids in a large number of different industrial processes, regardless of whether the volume of sample liquid transported into the process unit has to be discarded after enrichment and, if necessary, disposed of, or whether it can be returned to the process .

The target substance can be a biomolecule, for example. The biomolecule can be selected from the group consisting of: eukaryotic cells, components of eukaryotic cells, prokaryotic cells, components of prokaryotic cells, subcellular vesicles, bacteriophages, viruses or virus components, toxins, antibodies, nucleic acids and proteins.

For example, any aqueous solution can be used as a sample liquid, e.g. water and waste water samples, fermentation liquids, liquids from food technology, chemical, pharmaceutical or biochemical production processes.

The superabsorbent can comprise a plastic that absorbs water molecules to form a hydrogel. The super absorber can be, for example, one of the materials mentioned above. Although the plastic advantageously absorbs water or other polar solvent molecules, it does not absorb any biomolecules, in particular not the target substance. This is the case, for example, with the materials mentioned above and with the superabsorbent balls commercially available under the designations “water beads”, “aqua beads” or “water beads”. The superabsorbent can be used, for example, in the form of particles, e.g. as a powder, as granules or in the form of geometric bodies, in particular spheres. A suitable diameter of the particles, in particular of the spheres, is 100 to 5000 μm.

The generation of concentrated sample liquid can dosing the first initial volume of the sample liquid to that in the first Concentrator chamber submitted first amount of superabsorbent or dosing of the first amount of superabsorbent to the submitted in the first concentrator chamber first initial volume of the sample liquid.

The incubation of the mixture formed from the first initial volume of the sample liquid with the first quantity of the superabsorbent can be carried out over a predetermined, in particular selectable, period of time. In general, the increase in concentration of a target substance in the sample liquid that can be achieved within a period of time depends on the type of superabsorbent, the particle size of the superabsorbent, the size of the first amount of superabsorbent added, the temperature prevailing during incubation and the length of the selected period of time for the incubation. The period of time during which the incubation is carried out can be monitored by means of control electronics. In an alternative embodiment, it is also possible to monitor the volume of the liquid portion and to end the incubation when a desired, predetermined target volume is reached.

The temperature of the concentrator chamber and the mixture contained therein can be controlled during the incubation by means of a temperature control device of the concentrator unit. The temperature control device can be a heating and/or cooling device that can be operated automatically by means of control electronics for controlling and/or regulating a temperature of the mixture present in the concentrator chamber.

The method can further include the following step: after the incubation, removing at least part of the concentrated sample liquid from the first concentrator chamber. The removed liquid can be fed directly to a detection unit of an analysis device. Alternatively, as described below, the concentrated sample liquid can first be fed to a collection vessel. This allows the target substance to be concentrated in several initial liquid volumes taken as samples, e.g. from a process or a body of water, using the method described and combined for further detection. The pooled, concentrated samples can optionally be further concentrated again using the method described. This procedure is particularly advantageous when the concentration of the target substance in the original, untreated sample liquid is particularly low. The procedure is described in more detail below.

The sample liquid taken from the concentrator chamber and concentrated in a first stage can first be transported to a collection vessel. Subsequently, residues of the concentrated sample liquid and the first quantity of the super absorber can be removed from the concentrator chamber, for example by flushing the concentrator chamber with a flushing fluid, for example a flushing liquid or a flushing gas.

• - Dosieren und Transportieren eines zweiten initialen Volumens der Probeflüssigkeit über eine Flüssigkeitszuleitung in die erste Konzentratorkammer der Konzentrator-Einheit; und
• - Erzeugen von aufkonzentrierter Probeflüssigkeit durch Reduzieren des zweiten initialen Volumens der Probeflüssigkeit in der Konzentrator-Einheit und damit verbundenem Aufkonzentrieren von in dem zweiten initialen Volumen der Probeflüssigkeit enthaltenen Biomolekülen mittels einer zweiten Menge des Superabsorbers, wobei das Reduzieren und das damit verbundene Aufkonzentrieren durch Inkubieren eines aus dem zweiten initialen Volumen der Probeflüssigkeit mit der zweiten Menge des Superabsorbers gebildeten Gemisches erfolgt, und
• - Transportieren mindestens eines Teils der durch Reduzieren des zweiten initialen Volumens der Probeflüssigkeit erhaltenen aufkonzentrierten Probeflüssigkeit aus der ersten Konzentratorkammer in das Sammelgefäß.

Auf diese Weise können mehrere Chargen aufkonzentrierter Probeflüssigkeit erzeugt und im Sammelgefäß zusammengegeben werden. Dies ermöglicht es, eine sehr große Volumenmenge an Probe aufzukonzentrieren.The method can also include the following steps:

• - Dosing and transporting a second initial volume of the sample liquid via a liquid supply line into the first concentrator chamber of the concentrator unit; and
• - Generation of concentrated sample liquid by reducing the second initial volume of the sample liquid in the concentrator unit and the associated concentration of biomolecules contained in the second initial volume of the sample liquid using a second amount of superabsorbent, the reduction and the associated concentration by incubating a the mixture formed from the second initial volume of the sample liquid with the second quantity of the superabsorbent takes place, and
• - Transporting at least part of the concentrated sample liquid obtained by reducing the second initial volume of the sample liquid from the first concentrator chamber into the collection vessel.

In this way, several batches of concentrated sample liquid can be generated and combined in the collection vessel. This makes it possible to concentrate a very large volume of sample.

At least part of the concentrated sample liquid from the collection vessel can be dosed and transported into the first concentrator chamber or into a second concentrator chamber different from the first concentrator chamber for further concentration of the target substance by means of a third amount of superabsorbent. This further concentration is carried out in a completely analogous manner to that described for the first concentration, namely by creating a mixture of the concentrated sample liquid and the third quantity of the superabsorbent and incubating the mixture, which reduces the volume of the concentrated sample liquid again and the biomolecules contained, including the target substance, to be further concentrated. The further concentrated sample liquid thus obtained can be concentrated again in the same way or for further analysis can be fed directly to a detection unit of an analysis device. In this way, the sensitivity of the method can be further increased.

In all of its variants described above, the method can advantageously be carried out completely automatically by control electronics. For this purpose, the concentrator unit can have means for transporting liquids and for transporting the superabsorbent within the concentrator unit, which can include pumps and/or valves, for example.

• - Erzeugen von aufkonzentrierter Probeflüssigkeit aus mindestens einem ersten initialen Volumen der Probeflüssigkeit mittels des voranstehend beschriebenen Verfahrens zum Aufkonzentrieren mindestens einer biologischen Zielsubstanz in einer Probeflüssigkeit,
• - Transportieren mindestens eines Teils der erzeugten aufkonzentrierten Probeflüssigkeit aus der Konzentrator-Einheit in eine Detektionseinheit des Online-Analysegeräts, und
• - Qualitative oder quantitative Bestimmung der Zielsubstanz in der aufkonzentrierten Probeflüssigkeit und/oder in der Probeflüssigkeit, d.h. der ursprünglichen Probeflüssigkeit vor Aufkonzentrierung, mittels der Detektionseinheit und einer Steuerelektronik des Online-Analysegeräts.

The invention also relates to a method for automated online detection of at least one biological target substance in a sample liquid using an online analysis device. This procedure includes:

• - Generating concentrated sample liquid from at least a first initial volume of the sample liquid by means of the method described above for concentrating at least one biological target substance in a sample liquid,
• - Transporting at least part of the generated concentrated sample liquid from the concentrator unit into a detection unit of the online analysis device, and
• - Qualitative or quantitative determination of the target substance in the concentrated sample liquid and/or in the sample liquid, ie the original sample liquid before concentration, using the detection unit and control electronics of the online analysis device.

• - Freisetzen und/oder Isolieren von Nukleinsäuren aus den in der aufkonzentrierten Probeflüssigkeit enthaltenen Biomolekülen, insbesondere der mindestens einen biologischen Zielsubstanz, und Erzeugen einer die freigesetzten und/oder isolierten Nukleinsäuren umfassenden Lösung,
• - Amplifizieren mindestens einer in der Lösung enthaltenen Ziel-Nukleinsäure, und
• - Erfassen eines Messsignals, das eine von dem Fortschritt der Amplifizierung und/oder von einer Anzahl von Kopien der Ziel-Nukleinsäure abhängige Messgröße repräsentiert, wobei die Steuerelektronik des Analysegeräts anhand des erfassten Messsignals die qualitative oder quantitative Bestimmung der Zielsubstanz in der aufkonzentrierten Probeflüssigkeit und/oder in der Probeflüssigkeit vor Aufkonzentrierung durchführt. Die zu amplifizierende Ziel-Nukleinsäure kann entweder die zu detektierende Zielsubstanz oder eine Nukleinsäure der zu detektierenden Zielsubstanz sein.

The qualitative or quantitative determination can be carried out using molecular genetic methods.
In such an advantageous embodiment, the method further includes the following steps:

• - Releasing and/or isolating nucleic acids from the biomolecules contained in the concentrated sample liquid, in particular the at least one biological target substance, and generating a solution containing the released and/or isolated nucleic acids,
• - amplifying at least one target nucleic acid contained in the solution, and
• - Detection of a measurement signal, which represents a measurement variable that is dependent on the progress of the amplification and/or on a number of copies of the target nucleic acid, the control electronics of the analysis device using the measurement signal detected to determine the qualitative or quantitative determination of the target substance in the concentrated sample liquid and/or or in the sample liquid before concentration. The target nucleic acid to be amplified can be either the target substance to be detected or a nucleic acid of the target substance to be detected.

In an advantageous embodiment, the release and/or isolation of nucleic acids from the biomolecules contained in the concentrated sample liquid and the generation of the solution comprising the released and/or isolated nucleic acids are carried out in a first microfluidic unit of the detection unit. The solution is transported into a second microfluidic unit that can be fluidically connected to the first microfluidic unit, and the amplification and acquisition of the measurement signal is carried out in the second microfluidic unit. The first and the second microfluidic unit can be accommodated in two separate cartridges or in a common cartridge.

• - Versetzen der flüssigen Probe mit einem oder mehreren Lysereagenzien, um Nukleinsäuren der Biomoleküle freizusetzen,
• - Binden der freigesetzten Nukleinsäuren an ein Nukleinsäure bindendes Material; und
• - Waschen der gebundenen Nukleinsäuren mit einer Waschlösung, und
• - Ablösen der Nukleinsäuren von dem Nukleinsäure bindenden Material durch Eluieren.

Das hierbei erhaltene Eluat bildet in dieser Ausgestaltung die erwähnte, die freigesetzten und/oder isolierten Nukleinsäuren umfassende Lösung.The release and/or isolation of nucleic acids from the biomolecules contained in the concentrated sample liquid and the generation of the solution containing the released and/or isolated nucleic acids can include at least the following steps:

• - adding one or more lysis reagents to the liquid sample in order to release the nucleic acids of the biomolecules,
• - Binding of the released nucleic acids to a nucleic acid-binding material; and
• - washing the bound nucleic acids with a washing solution, and
• - Detachment of the nucleic acids from the nucleic acid-binding material by elution.

In this embodiment, the eluate obtained in this way forms the mentioned solution comprising the released and/or isolated nucleic acids.

• - thermisches Behandeln der in der aufkonzentrierten Probeflüssigkeit enthaltenen Biomoleküle in einer Pufferlösung, beispielsweise in einer phosphatgepufferten Salzlösung, in Wasser, in einem Tris-Puffer oder in einem Puffer, der eine geringe Konzentration an einem Detergenz enthält. In diesem Fall bildet die Pufferlösung mit den durch die thermische Behandlung freigesetzten Nukleinsäuren die zuvor erwähnte, die freigesetzten und/oder isolierten Nukleinsäuren umfassende Lösung.

In an alternative embodiment, the release and/or isolation of nucleic acids from the biomolecules contained in the concentrated sample liquid and the generation of the solution containing the released and/or isolated nucleic acids can include the following steps:

• - Thermal treatment of the biomolecules contained in the concentrated sample liquid in a buffer solution, for example in a phosphate-buffered saline solution, in water, in a Tris buffer or in a buffer containing a low concentration of a detergent. In this case, the buffer solution with the nucleic acids released by the thermal treatment forms those previously mentioned solution comprising the released and/or isolated nucleic acids.

Thermal release may involve dispersing the sample in the buffered solution and incubating. This variant of the method is suitable, for example, for applications in which the target substance to be determined is a nucleic acid that is already freely present in the liquid to be analyzed. In this alternative embodiment of the method, a classic lysis can therefore be omitted. It is also possible to use the method when the target substance is a bacterium or a virus as a biomolecule. In this case, the biomolecules contained in the sample can be thermally/chemically destroyed and the nucleic acid contained can be released.

The amplification carried out, for example, in the second microfluidic unit can be carried out, for example, using conventional PCR-based methods. The qualitative or quantitative determination of the target substance can also be carried out in a conventional manner by using a sensor to record at least one measured value of a measured variable at a specific point in time or at a plurality of points in time, which corresponds to the number of values present at the respective point in time due to the amplification generated copies of a target nucleic acid. From the measured value obtained or the course of measured values as a function of time, the presence of the target nucleic acid and thus the biological target substance in the sample liquid can be qualitatively detected or a quantitative value, e.g. a concentration, of the biological target substance in the original sample liquid or in the sample liquid after concentration using the determine the method described above and output it as a measurement result from the control electronics.

All steps of the method according to the invention described here for online detection of at least one biological target substance in a sample liquid in all specified configurations can be carried out automatically using the control electronics of the online analysis device. For this purpose, it can, for example, transport liquid (sample liquid and/or reagents) or transport solids (superabsorber material) by actuating controllable valves and/or pumps and control the incubation time and temperature when concentrating and/or releasing nucleic acids by means of a predetermined sequence program and/or regulate.

• - eine erste Konzentratorkammer;
• - mindestens eine in die erste Konzentratorkammer mündende Flüssigkeitszuleitung; und
• - mindestens eine in die erste Konzentratorkammer mündende Flüssigkeitsableitung,

wobei die Konzentrator-Einheit dazu eingerichtet ist, eine erste Menge eines Superabsorbers zu einem über die Flüssigkeitszuleitung in die erste Konzentratorkammer eingeleiteten initialen Flüssigkeitsvolumen der Probeflüssigkeit zuzugeben, oder dazu eingerichtet ist, das initiale Flüssigkeitsvolumen zu der ersten Menge des Superabsorbers zuzugeben.The invention also includes a concentrator unit for the automated implementation of a concentration of a target substance in a sample liquid, in particular according to the method described above, comprising:

• - a first concentrator chamber;
• - at least one liquid feed line opening into the first concentrator chamber; and
• - at least one liquid discharge line opening into the first concentrator chamber,

wherein the concentrator unit is set up to add a first amount of a super absorber to an initial liquid volume of the sample liquid introduced into the first concentrator chamber via the liquid supply line, or is set up to add the initial liquid volume to the first amount of super absorber.

In one possible configuration, the first amount of superabsorbent can be placed in the first concentrator chamber. In this configuration, the concentrator chamber can be configured as an exchangeable cartridge. When using this configuration, the replaceable cartridge with the super absorber can be exchanged for a new cartridge for the concentration of the target substance in each new batch of the sample liquid.

In an alternative configuration, the concentrator unit can have at least one reservoir or be connected to a reservoir containing superabsorbent, in particular in the form of particles. One of the materials mentioned further above, in particular in spherical form, is preferably used as the superabsorbent. Commercially available super absorber beads with a diameter of the order of magnitude of 1 mm, which are also commercially available under the name “water beads”, “aqua beads”, “water beads” or “gel beads”, have proven to be very advantageous. Super absorber spheres of this size can be dispersed very well in the concentrator chamber.

The reservoir can be connected to the first concentrator chamber, for example, via a superabsorbent supply line that can be closed with a valve that can be controlled in particular by means of control electronics, such that when the valve is open, at least part of the superabsorbent contained in the reservoir can flow into the first as a first quantity of the superabsorbent reached the concentrator chamber. The transport of superabsorbent can be effected by gravity. However, it is also possible for the concentrator unit to have means for transporting the superabsorbent present, for example, in the form of particles, eg a pump and/or means for generating a gas flow which transports the superabsorbent particles. For dosing and transport of the superabsorbent can a control electronics designed for the automatic operation of the concentrator unit are used, which are set up to actuate the mentioned means and/or the mentioned valve in order to meter the first quantity of superabsorbent and to transport it into the concentrator chamber. These control electronics can be set up accordingly in interaction with pumps and/or valves to meter the initial volume of the sample liquid into the concentrator chamber.

The concentrator device can have means for moving, in particular stirring, the liquid contained in the reaction chamber. The means can include, for example, a stirrer, a feed line for an inert gas into the reaction chamber and a discharge line for the inert gas, which are arranged such that the inert gas flows through the liquid contained in the reaction chamber, or have a drive for moving the reaction chamber.

The concentrator unit can also have a temperature control device, which is designed to control the temperature of the first concentrator chamber. The temperature control device can have heating and/or cooling elements, for example, which can be operated by means of the control electronics in order to control and/or regulate a temperature of a liquid held in the concentrator chamber.

The liquid discharge line for discharging at least part of the liquid volume of the sample liquid present in the first concentrator chamber can be fluidically connectable to a collecting vessel.

In a further configuration, the collection vessel can have a collection vessel outlet line which can be fluidically connected to the first concentrator chamber. In this way, sample liquid in the collection vessel that has already been concentrated in a first stage can be fed back into the first concentrator chamber in order to be further concentrated in a second stage by again feeding superabsorbent to the concentrated sample liquid in the first concentrator chamber and incubating the mixture.

In an alternative embodiment, the concentrator unit can comprise an additional second concentrator chamber, with the liquid discharge line of the first concentrator chamber connecting it to the second concentrator chamber, in particular via a collecting vessel; and a further liquid discharge line opening into the second concentrator chamber,
wherein the concentrator unit is set up to add a second amount of the super absorber to a volume of the sample liquid concentrated in the first concentrator chamber introduced into the second concentrator chamber or to add the liquid volume to the second amount of super absorber.

The invention also includes an online analysis device for detecting at least one biological target substance in a sample liquid, in particular according to the method described above. The analyzer includes: control electronics;
a concentrator unit according to one of the configurations described above, wherein the concentrator unit has a liquid outlet for concentrated sample liquid produced from the sample liquid in the concentrator unit; and
a detection unit that can be fluidically connected to the liquid outlet of the concentrator unit; and at least one transport device that can be controlled by the control electronics, e.g. a pump, which is set up to transport concentrated sample liquid from the liquid outlet of the concentrator unit to the detection unit,
wherein the control electronics are set up to use the detection unit to qualitatively or quantitatively determine the target substance in the concentrated sample liquid and/or in the sample liquid before concentration based on a molecular genetic method.

The online analysis device can also have a sample pump, with the control electronics being set up to control the sample pump to transport a specified volume of the sample liquid into the concentrator unit. The sample feed line can be fluidly connectable to a sample template or directly to a body of water or a line in a process plant, a fermenter or another process container.

• - eine mit einem Flüssigkeitsausgang für aufkonzentrierte Probenflüssigkeit der Konzentrator-Einheit fluidisch verbindbare erste Mikrofluidikeinheit, die dazu eingerichtet ist, aus in der aufkonzentrierten Probeflüssigkeit enthaltenen Biomolekülen, insbesondere der mindestens einen biologischen Zielsubstanz, Nukleinsäuren freizusetzen und/oder zu isolieren,
• - eine mit der ersten Mikrofluidikeinheit verbundene zweite Mikrofluidikeinheit, die dazu eingerichtet ist, ein die freigesetzten und/oder isolierten Nukleinsäuren umfassendes Eluat aufzunehmen und eine Ziel-Nukleinsäure zu amplifizieren; und
• - einen, insbesondere optischen, Sensor, der dazu eingerichtet ist, ein von dem Fortschritt einer in der zweiten Mikrofluidikeinheit durchgeführten Amplifizierung und/oder von einer Anzahl von Kopien der Ziel-Nukleinsäure in der zweiten Mikrofluidikeinheit abhängiges Messsignal zu erzeugen und an die Steuerelektronik auszugeben; und

wobei die Steuerelektronik dazu eingerichtet ist, anhand des von dem Sensor ausgegebenen Messsignals die biologische Zielsubstanz in der Flüssigkeit qualitativ und/oder quantitativ zu bestimmen.The detection unit can have the following components:

• - a first microfluidic unit which can be fluidically connected to a liquid outlet for concentrated sample liquid of the concentrator unit and is set up to release and/or isolate nucleic acids from biomolecules contained in the concentrated sample liquid, in particular the at least one biological target substance,
• - a second microfluidic unit which is connected to the first microfluidic unit and is set up to receive an eluate comprising the released and/or isolated nucleic acids and to amplify a target nucleic acid; and
• a sensor, in particular an optical sensor, which is set up to generate a measurement signal dependent on the progress of an amplification carried out in the second microfluidic unit and/or on a number of copies of the target nucleic acid in the second microfluidic unit and to output it to the control electronics; and

wherein the control electronics are set up to qualitatively and/or quantitatively determine the biological target substance in the liquid based on the measurement signal output by the sensor.

The liquid outlet of the concentrator unit can be fluidically connectable to the aforementioned first concentrator chamber and/or to a second concentrator chamber that may be present and/or to a collection vessel that is also optionally present. In various design or operating alternatives of the online analysis device, concentrated sample liquid can thus be transported directly from the first or the optionally available second concentrator chamber or via the optionally available collection vessel into the detection unit.

The first and the second microfluidic unit can be arranged together in one, in particular replaceable, cartridge. Alternatively, the first microfluidic unit can be arranged in a first cartridge and the second microfluidic unit can be arranged in a second cartridge that is different from the first cartridge, the first and second cartridges being fluidically connectable to one another in order to transport liquid from the first cartridge into the second . The first and the second cartridge can be designed to be exchangeable.

The first microfluidic unit can be set up to take up the concentrated sample with the biomolecules it contains and to add one or more lysis reagents to release the nucleic acid of the biomolecules contained in the sample, then absorb the released nucleic acids, if necessary with the addition of a binding of the nucleic acid to a To bind nucleic acid-binding material enhancing / mediating component and to wash the nucleic acids bound to this material one or more times with a washing solution. The control electronics can be set up to control a transport of the particles and optionally the reagents through the first microfluidic unit.

The first microfluidic unit can also be set up to detach the nucleic acids from the nucleic acid-binding material by elution and to transport the eluate into the second microfluidic unit via a fluid line connecting the first microfluidic unit to the second microfluidic unit. The control electronics can be set up to control the elution and the transport of the eluate.

In an alternative embodiment, the first microfluidic unit can be set up to take up the concentrated sample with the biomolecules it contains and to absorb the biomolecules in a buffer solution, for example in a phosphate-buffered saline solution (PBS buffer), water or a Tris buffer, or optionally a buffer containing a detergent, thermally/chemically released, the control electronics being set up to control the thermal release of the biomolecules and to transport the buffer solution with the biomolecules or nucleic acids dissolved therein into the second microfluidic unit for the subsequent amplification. This configuration is suitable, for example, for use in an application in which the biological target substance to be detected is a nucleic acid that is freely present in the liquid.

The first microfluidic unit can contain the reaction components required for releasing and/or isolating the nucleic acid in solid form, for example in the form of pellets obtained by lyophilization, or in reagent chambers closed by microvalves or in reagent packs closed by foils, which are automated by the action of force or heat, in particular by means of the control electronics, can be opened.

Reagents for the amplification can be stored in a corresponding manner in the second microfluidic unit. It can also contain one or more detection chambers, in which the amplification of a target nucleic acid or else several different target nucleic acids can be carried out in parallel. The second microfluidic unit can include a temperature control device for the detection chambers. The temperature control device can, for example, have thermoelectric elements that can be controlled by the control electronics.

The control electronics described here and above in connection with the method according to the invention and the concentrator unit according to the invention can have an electronic data processing device with at least one processor and a memory in which one or more operating programs are stored which can be executed by means of the processor in order to optimize the analysis device to control automatically and to determine qualitative or quantitative analysis results from the measurement signals of the sensor. The operating programs can be designed in such a way that the electronic control unit controls the analysis device for carrying out the method described above. The control electronics can also have a user interface, for example a touch screen or another display in combination with an input keyboard. The control electronics can be set up to be connected wirelessly by radio or via a data line to another data processing device, eg a computer, a process controller, a particularly portable display or operating device for communication.

The automatic control of the concentration carried out in the concentrator unit can be carried out by means of the same control electronics that are also used to control the other components of the analysis device. Alternatively, the concentrator unit can also have its own electronic control system, which is set up to carry out the concentration of the sample liquid automatically in the manner described. In this embodiment, the control electronics of the concentrator unit are advantageously connected for communication to the control electronics of the analysis device, which control the other components of the analysis device, namely in particular the detection unit.

The analyzer can thus perform on-line determinations of biological target substances in a liquid without requiring manual sampling or preparation or transport of samples to a laboratory. In particular, the analysis device can carry out sampling and detection cycles fully automatically, which can be event-controlled by the electronic control system or carried out at predetermined time intervals, for example.

• 1 eine schematische Darstellung eine Online-Analysegeräts zur qualitativen oder quantitativen Bestimmung einer biologischen Zielsubstanz in einer Flüssigkeit;
• 2 ein erstes Ausführungsbeispiel einer Konzentrator-Einheit des in 1 dargestellten Online-Analysegeräts nach einem ersten Ausführungsbeispiel; und
• 3 ein zweites Ausführungsbeispiel einer Konzentrator-Einheit für ein Online-Analysegerät.

The invention is described below with reference to the exemplary embodiments illustrated in the figures. The same reference symbols designate the same components of the components shown in the figures. Show it:

• 1 a schematic representation of an online analysis device for the qualitative or quantitative determination of a biological target substance in a liquid;
• 2 a first embodiment of a concentrator unit of the in 1 illustrated online analysis device according to a first embodiment; and
• 3 a second embodiment of a concentrator unit for an online analysis device.

In 1 an exemplary embodiment of an online analysis device 1 for the detection of a biological target substance in a sample liquid is shown schematically. In the present example, the analysis device 1 has a housing 2 in which all the components of the device are accommodated. These components include, in detail, a concentrator unit 3, a detection unit 4 and control electronics 5. The concentrator unit 3 enables the detection of biomolecules contained in a sample liquid even in very low concentrations, since it is designed to contain biological target molecules in a volume of a to concentrate the sample liquid to be analyzed and to forward the concentrated sample liquid to the detection unit 4 for detection and, if necessary, preparatory steps such as extraction and/or isolation of nucleic acids.

The concentrator unit 3 is fluidly connected via a sample feed line 6 to a sample receiver 7 or to a process container, e.g. a pipeline or a reactor or a fermenter, in which the liquid to be analyzed is contained. A pump 9, which can be controlled by the electronic control unit 5 according to an operating program stored in the electronic control unit 5 and executed by it, is used to transport and meter the liquid from the sample receiver 7 into a first concentrator chamber 8 contained in the concentrator unit 3. The first concentrator chamber 8 can also be fluidically connected to reservoirs 10, 11. The reservoirs 10, 11 contain substances which serve to enrich the biomolecules contained in the liquid. In the present example, each reservoir 10, 11 is fluidically connected to the concentrator chamber in the concentrator unit 3 via a fluid line. A pump 12 , 13 is used to transport the substances contained in the reservoirs through the fluid lines. It is also possible for the reservoirs 10 , 11 to be integrated directly into the concentrator unit 3 . The substances can of course be transported by other means known to those skilled in the art, e.g. pneumatically or by utilizing gravity or a pressure, e.g. hydrostatic.

The concentrator unit 3 can optionally have a temperature control unit (not in 1 shown), which is used to set a specific temperature in the concentrator chamber 8. The temperature control unit can include resistance heating, cooling and/or thermoelectric elements for selective heating or cooling. It can be connected to the control electronics 5 , the control electronics 5 being set up to control or regulate the temperature in the concentrator chamber 8 .

The concentrator unit 3 may also include a device 14 for agitation or stirring have a liquid or liquid mixture contained in the reaction chamber 8 . In the present example, the device 14 comprises a drive for moving, for example shaking, the reaction chamber 8 . The device 14 , in particular the drive, can be controlled by the control electronics 5 .

The concentrator chamber 8 can be fluidically connected to the detection unit 4 or to a liquid outlet 17 via the fluid line 15 either by means of a valve 16 that can be actuated by the control unit 5 .

In the present example, the detection unit 4 has an analysis cartridge in which two microfluidic units fluidically connected to one another via the fluid line 18, namely a first microfluidic unit 19 and a second microfluidic unit 20, are integrated. In another embodiment, the microfluidic units can also be accommodated in separate cartridges. In the present example, the analysis cartridge can be replaced.

The first microfluidic unit 19 is set up to receive a sample containing the concentrated biomolecules from the reaction chamber and to prepare it for subsequent amplification and detection in the second microfluidic unit 20 . This can be done by means of extraction and isolation processes known per se. For this purpose, the first microfluidic unit 19 includes reagents and optionally a temperature control unit for incubating reaction mixtures produced in the microfluidic unit 19 . In order to control a transport of the sample through the first microfluidic unit 19 and to carry out the extraction and/or isolation of nucleic acids, the detection unit 4 comprises suitable means known to the person skilled in the art, which can be controlled by the control electronics 5 . The second microfluidic unit 20 includes means for amplification, i.e. reagents and temperature control elements, e.g. thermoelectric elements, as well as means for transporting and aliquoting fluids within the second microfluidic unit 20. For detecting a progress of the amplification in a manner known per se, e.g. by means of a real time PCR-based method, the detection unit has a sensor 21 which can, for example, carry out fluorescence measurements in the second microfluidic unit 20 and can output measurement signals to the control electronics 5 . The control electronics 5 are set up to determine a qualitative or quantitative analysis result based on the measurement signals.

The control electronics 5 can be a central processing unit, e.g. a CPU with a processor and memory and operating programs stored therein. It can also be divided among several computing units within the analysis device, e.g. the concentrator unit 3 and the detection unit 4 can each have their own on-site electronics, which are connected to higher-level electronics for communication, with the higher-level electronics and the on-site electronics together form the control electronics 5.

In 2 is a first embodiment of a concentrator unit 3 of the in 1 illustrated online analysis device 1 shown in detail. the inside 2 shown structure of the concentrator unit 3 is only one possible embodiment. A person skilled in the art can find numerous variants without departing from the spirit of the invention.

The concentrator unit 3 has a concentrator chamber 8 with various inlets and outlets. The feed line 23 is connected to a storage container which serves as a reservoir 10 and contains a super absorber 24, e.g. in the form of balls made of the super absorber material. The super absorber 24 is added to the concentrator chamber 8 via the feed line 23. A first opening 25 serves to feed in the sample liquid. A specific initial volume of the sample liquid is fed into the concentrator chamber 8 via it. A second opening 26 is connected to the fluid line, which connects the reaction chamber (1) to a sample outlet and to the detection unit. The second opening 26 can have a diameter that is smaller than the diameter of the super absorber spheres, so that only the liquid portion of the mixture of sample liquid and adsorber can be drained from the concentrator chamber 8 via the second opening 26 in the direction of the detection unit 4 . Optionally, the second opening 26 can also have a size selector, e.g.

The super absorber contained in the concentrator chamber 8 and residues of the sample liquid can be removed from the concentrator chamber 8 through a third opening 27 serving as a drain, which in the present example can be introduced into the concentrator chamber 8 via the sample liquid opposite the opening 25 . A rinsing liquid or a rinsing gas can be passed through the concentrator chamber 8 in order to completely remove the super absorber 24 and the residues of the sample liquid. In the present example, the rinsing liquid can either be the sample liquid that is conducted from the sample holder 7 through the concentrator chamber 8 . Alternatively, the rinsing liquid can be a liquid stored in the reservoir 11 . In the latter case, the reservoir 11 is fluidically connected to the first opening 25 . Optional are in the respective Valves are arranged in the supply lines, which can be actuated by the control electronics 5.

• 1) Einleiten eines ersten initialen Probeflüssigkeitsvolumens aus der Probevorlage 7 in die Konzentratorkammer 8,
• 2) Zugeben einer ersten Menge des Superabsorbers 24 aus dem Reservoir 10 in die Konzentratorkammer 8 zu dem ersten initialen Probeflüssigkeitsvolumen,
• 3) Inkubieren des Gemisches aus dem Superabsorber 24 und der Probeflüssigkeit während einer von der Steuerelektronik 5 gemessenen Zeitspanne, dabei reduziert sich das Volumen des flüssigen Anteils des Gemisches unter Aufkonzentrierung von Biomolekülen, insbesondere einer zu bestimmenden biologischen Zielsubstanz in dem flüssigen Anteil,
• 4) Nach Ablauf der Zeitspanne für die Inkubation Ableiten des flüssigen Anteils.

The control electronics 5 can include an operating program that it can execute to carry out the following procedure:

• 1) introducing a first initial sample liquid volume from the sample receiver 7 into the concentrator chamber 8,
• 2) adding a first quantity of the superabsorbent 24 from the reservoir 10 into the concentrator chamber 8 to the first initial sample liquid volume,
• 3) Incubating the mixture of the super absorber 24 and the sample liquid for a period of time measured by the control electronics 5, the volume of the liquid portion of the mixture is reduced with concentration of biomolecules, in particular a biological target substance to be determined in the liquid portion,
• 4) At the end of the incubation period, drain the liquid portion.

As already described above, the super absorber absorbs polar solvents, e.g. water in the present embodiment, from the sample liquid to form a gel. In contrast, biomolecules, in particular the biological target substance to be determined, are not absorbed by the superabsorber. During the incubation, the volume of the liquid portion of the mixture of superabsorbent and sample liquid is thus reduced, and the target substance in the liquid is concentrated. The incubation time can be selected in such a way that it is ensured that the concentration of the target substance in the concentrated sample liquid after the incubation is above a threshold value, e.g. above a detection limit specified by the detection unit. The length of time until the desired reduction in volume or the desired concentration depends on the following factors: type of superabsorbent, particle size of the superabsorbent particles, amount of superabsorbent added, temperature during incubation. A quantity of super absorber and incubation time suitable for a specific type of sample liquid can be determined by means of preliminary tests and stored in the control electronics.

The control unit 5 can be designed to set or regulate the temperature of the liquid contained in the concentrator chamber 8 or of the mixture of sample liquid and superabsorbent to a constant value by means of the previously mentioned temperature control device. All of these steps can be carried out automatically using the control unit 5 . The liquid part is a concentrated sample liquid, i.e. sample liquid that contains the biological target substance in an increased concentration due to the volume reduction caused by the absorption of water by the superabsorbent. In a possible configuration according to the exemplary embodiment described here, the concentrated sample liquid can be transported directly into the detection unit 4 . With the method described here and the concentrator unit 3 described here for carrying out this method, it is possible to reduce a large-volume water sample to a much smaller sample volume and correspondingly to achieve a large increase in the concentration of biomolecules. This facilitates and/or accelerates subsequent detection using molecular genetic methods. If the concentration of the biomolecules in the sample liquid is originally very low, the method described here can even make molecular-genetic detection of the target substance possible in the first place.

A further advantage results from the fact that it is possible with the approach according to the invention to concentrate both bacteria, viruses and free nucleic acids in a completely unselective manner. Accordingly, it is also possible in a very simple manner to first concentrate and extract several different biological target substances with the method and then to determine them using a PCR-based method.

The opening 26 of the concentrator unit 3 is fluidically connected to the fluid line 15 of the analysis device 1 , which can be connected via the valve 16 to the detection unit 4 on the one hand and to the liquid outlet 17 on the other hand. The detection of the target substance is described in more detail below: The concentrated sample liquid is transported through the fluidic line 15 into the first microfluidic unit 19 via the fluidic line 15 by means of the control electronics 5 . A further pump, which is controlled by the control electronics 5, can be used for this purpose. The valve 16 is also actuated by the control electronics 5 . Automated nucleic acid extraction and/or isolation takes place in the first microfluidic unit 19, and automated amplification and detection by means of PCR or real-time PCR takes place in the second microfluidic unit 20 connected downstream of the first microfluidic unit 19. Microfluidic units suitable for the method described here for automated nucleic acid extraction, amplification and detection are basically known in the prior art. A centrifugal platform is advantageous for the present application, as is the case, for example, in WO 2013/045631 A1 and EP 2 621 632 A1 is described, but it is also possible to use other microfluidic platforms or lab-on-chip systems that have one Use capillary forces, pumps, or pneumatics to transport liquids and reagents rather than centrifugal force.

The nucleic acids are extracted and/or isolated in the first microfluidic unit 19 using known reagents and methods, as already described in the introduction. If the biological target substance is not already freely present in the liquid taken from the sample, the biomolecules contained in the concentrated sample liquid are lysed. The free nucleic acids are bound to a nucleic acid-binding material, e.g. in particle form. The nucleic acid-binding material with the nucleic acids bound to it is then washed in a known manner and the nucleic acids are finally detached from the material. The eluate containing the dissolved nucleic acids is transported into the second microfluidic unit 20, again controlled by the control electronics 5.

In the second microfluidic unit 20, specific volumes of the eluate are transported into one or more detection chambers and amplification reagents are added. An amplification then takes place in the detection chambers, the progress of which is monitored by means of fluorescence measurements with the sensor 21 . The sensor 21 outputs measurement signals to the control electronics 5 . From the measurement signals, this determines either a qualitative value that indicates whether the specific biological target substance is contained in the liquid taken from the sample holder 7 . Alternatively or additionally, the control electronics 5 can also determine a quantitative value from the measurement signals, which represents a concentration of the biological target substance in the liquid. In an advantageous embodiment, different target nucleic acids can be amplified in a number of detection chambers and a number of different biological target substances in the liquid can thus be determined qualitatively or quantitatively in parallel.

Before or after the transfer of the concentrated liquid from the concentrator unit 3 to the detection unit 4 or after detection has taken place, the liquid remaining in the concentrator unit 3 is discharged from the process unit 3 through the liquid outlet 17 . The concentrator unit 3 can be sufficiently rinsed with a rinsing medium, for example with liquid sucked in from the sample receiver 7, and the process can now be repeated for a further measurement. The microfluidic units 19, 20 in the detection unit can be replaced by new microfluidic units 19, 20 for further measurement. All of the steps described here can be carried out automatically by the control electronics 5 .

In 3 is a schematic representation of an alternative embodiment of a concentrator unit 33, which instead of in 2 shown concentrator unit 3 in the analyzer 1 can be used. The concentrator unit 33 is set up to concentrate a sample liquid in several stages and thus allows the concentration of even target substances that are only present in an extremely low concentration in the original sample liquid. The concentrator unit 33 has a first concentrator chamber 8, which has an inlet 25 for sample liquid, a supply line 23 for super absorbers, eg in the form of balls with a diameter of the order of 1 mm, and two discharge lines 26,27. The feed line 23 for the superabsorbent can be connected to a reservoir 10 in which a supply of superabsorbent is contained. The first derivation 26 from the concentrator chamber 8 leads to a collection vessel 28, the second derivation 27 serves as a derivation for used liquid and used superabsorbent and can be connected, for example, to a waste collection container.

The collection vessel 28 can be fluidically connected to a second concentrator chamber 30 via a liquid discharge line 29 . The second concentrator chamber 30 can be designed identically to the first concentrator chamber 8. In the present example, it has a further feed line 31 for superabsorbents, which can be fluidically connected to the same reservoir 10 of the analysis device as the feed line 23 to the first concentrator chamber 8. The second concentrator chamber has a first derivative 15 which can be fluidically connected to the detector unit 4 of the analysis device in order to supply the detector unit with concentrated sample liquid for the qualitative and/or quantitative detection of the biological target substance. The concentrator chamber 8 has a second drain line 32 for draining used liquid and/or used superabsorbent.

A multi-stage concentration of a target substance in a sample liquid can be carried out using the concentrator unit 33, in particular automatically using control electronics, e.g. the control electronics 5 of the analysis device, in the following way: In a first stage, a first initial volume of the to analyzing sample liquid are introduced. After addition of a first quantity of the superabsorbent, the mixture thus formed can be incubated for a predetermined period of time or until a certain final volume of the liquid portion is reached. At least part of the after the Ink The remaining liquid portion can then be routed from the concentrator chamber 8 into the collection vessel 28 via the first discharge line 26 . Residual liquid and used super absorber can be discharged from the concentrator chamber via the discharge line 27 .

In the same way, one or more additional initial volumes of the sample liquid in the first concentrator chamber 8 can be reduced in order to concentrate the target substance in these additional volumes. After the respective incubation, at least part of the respectively remaining liquid portion is correspondingly transferred into the collection vessel 28 .

The collected sample liquid, which has already been concentrated in a first stage, can be further concentrated in a second stage by means of the second concentrator chamber 30 . For this purpose, at least part of the concentrated sample liquid contained in the collection vessel 28 can be introduced into the second concentrator chamber 30 via the outlet line 29 of the collection vessel 28 . A second amount of superabsorbent is introduced into the second concentrator chamber 30 via the feed line 31 for superabsorbent. The mixture thus formed is incubated in the second concentrator chamber 30 for a predetermined period of time and/or until a desired volume of the liquid portion is reached. At least part of the liquid portion can then be fed via the liquid line 15 to the detector unit 4 of the analysis device. Liquid that is not required and/or the used super absorber can be discharged from the second concentrator chamber 30 via the discharge line 32 .

All the steps of the method described here can be carried out fully automatically by control electronics, for example the control electronics 5 of the analysis device 1 . For this purpose, the concentrator unit has suitable means for transporting liquid and superabsorbent particles through the fluid lines into the various chambers, in particular pumps and valves (not in 3 shown), which actuates the control electronics for carrying out the described method for concentrating the target substance in the sample liquid.

The analysis device and method according to the invention thus ideally solves the task by linking available device systems for detection and enables online liquid analysis of biological targets to be carried out, which is particularly advantageous for monitoring water or waste water in networks, sewage treatment plants or treatment plants as well as water bodies appears. The method according to the invention makes it possible for the first time to implement molecular genetic monitoring of biological targets in a practical and economical manner without manual intervention, starting with a large-volume sample up to detection.

QUOTES INCLUDED IN DESCRIPTION

This list of documents cited by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent Literature Cited

• US 2015/0224502 A1 [0005, 0006]
• WO 2013/045631 A1 [0076]
• EP 2621632 A1 [0076]

Claims (32)

Method for concentrating at least one biological target substance in a sample liquid, comprising: - Dosing and transporting a first initial volume of the sample liquid via a liquid supply line (25) into a first concentrator chamber (8) of a concentrator unit (3, 33); and - Generation of concentrated sample liquid by reducing the first initial volume of the sample liquid in the concentrator unit (3, 33) and the associated concentration of the at least one biological target substance contained in the first volume of the sample liquid by means of a first quantity of a superabsorbent (24), the Reducing and the associated concentration is carried out by incubating a mixture formed from the first initial volume of the sample liquid with the first quantity of the superabsorbent (24). procedure after claim 1 , wherein the at least one biological target substance is a biomolecule. procedure after claim 1 or 2 , wherein the super absorber (24) comprises a plastic that absorbs water molecules to form a hydrogel. procedure after claim 3 , whereby the plastic does not absorb any biomolecules, especially not the target substance. Procedure according to one of Claims 1 until 4 , wherein the super absorber (24) is used in the form of particles, for example as a powder, as granules or in the form of geometric bodies, in particular spheres. Procedure according to one of Claims 1 until 5 , wherein the production of concentrated sample liquid includes the dosing of the first initial volume of the sample liquid to the first quantity of superabsorbent (24) provided in the first concentrator chamber (8) or the dosing of the first quantity of superabsorbent (24) to the quantity in the first concentrator chamber ( 8) submitted first initial volume of sample liquid. Procedure according to one of Claims 1 until 6 , wherein the incubation of the mixture formed from the first initial volume of the sample liquid with the first quantity of the superabsorbent is carried out over a predetermined, in particular selectable, period of time. Procedure according to one of Claims 1 until 7 , wherein the concentrator chamber (8) and the mixture contained therein is heated during incubation by means of a temperature control device of the concentrator unit (3, 33). Procedure according to one of Claims 1 until 8th , further comprising: after the incubation, removing at least part of the concentrated sample liquid from the first concentrator chamber (8). procedure after claim 9 , the concentrated sample liquid taken from the concentrator chamber (8) being transported into a collecting vessel (28). procedure after claim 10 , further comprising: - dosing and transporting a second initial volume of the sample liquid via a liquid supply line (25) into the first concentrator chamber (8) of the concentrator unit (33); and - generating concentrated sample liquid by reducing the second initial volume of the sample liquid in the concentrator unit (33) and the associated concentration of biomolecules contained in the second initial volume of the sample liquid by means of a second quantity of the superabsorbent, the reduction and the associated Concentration takes place by incubating a mixture formed from the second initial volume of the sample liquid with the second quantity of the super absorber, and - transporting at least part of the concentrated sample liquid from the first concentrator chamber (8) into the collection vessel (28). procedure after claim 11 , wherein at least a part of the concentrated sample liquid from the collection vessel (28) for further concentration by means of a third quantity of the super absorber (24) into the first concentrator chamber (8) or into a second concentrator chamber (30 ) is metered and transported. Procedure according to one of Claims 1 until 12 , wherein the method is automated by control electronics (5) is carried out. Method for the automated online detection of at least one biological target substance in a sample liquid using an online analysis device (1), comprising: - generating concentrated sample liquid from at least a first initial volume of the sample liquid using the method according to one of Claims 1 until 13 - Transporting at least part of the generated concentrated sample liquid from the concentrator unit (3) into a detection unit (4) of the online analysis device, - Qualitative or quantitative determination of the target substance in the concentrated sample liquid and/or in the sample liquid before concentration using the detection unit (4) and control electronics (5) of the online analysis device (1). procedure after Claim 14 , wherein the method further comprises: - releasing and/or isolating nucleic acids from the biomolecules contained in the concentrated sample liquid and generating a solution comprising the released and/or isolated nucleic acids, - amplifying at least one target nucleic acid contained in the solution, and - Detection of a measurement signal that represents a measurement variable that is dependent on the progress of the amplification and/or on the number of copies of the target nucleic acid, the control electronics (5) of the analysis device (1) using the measurement signal detected to carry out the qualitative or quantitative determination of the target substance in of the concentrated sample liquid and/or in the sample liquid before concentration. procedure after claim 15 , wherein the release and/or isolation of nucleic acids from the biomolecules contained in the concentrated sample liquid and the generation of the solution comprising the released and/or isolated nucleic acids is carried out in a first microfluidic unit (19) of the detection unit, the solution in one with the first Microfluidic unit (19) fluidically connectable second microfluidic unit (20) is transported, and amplifying and detecting the measurement signal in the second microfluidic unit (20) is carried out. Procedure according to one of Claims 15 or 16 , wherein the release and/or isolation of nucleic acids from the biomolecules contained in the concentrated sample liquid and the generation of the solution comprising the released and/or isolated nucleic acids comprises at least the following steps: - adding one or more lysis reagents to the liquid sample in order to to release biomolecules, - binding of the released nucleic acids to a nucleic acid-binding material; - washing the bound nucleic acids with a washing solution, and - detaching the nucleic acids from the nucleic acid-binding material by elution. Procedure according to one of Claims 15 or 16 , wherein the release and/or isolation of nucleic acids from the biomolecules contained in the concentrated sample liquid and the generation of the solution comprising the released and/or isolated nucleic acids comprises the following steps: thermal treatment of the biomolecules contained in the concentrated sample liquid in a buffer solution, for example in a phosphate buffered saline solution, in water, in a Tris buffer or in a buffer containing a low concentration of a detergent. Procedure according to one of Claims 14 until 18 , whereby the control electronics of the online analysis device carry out all process steps automatically. Concentrator unit (3, 33) for the automated implementation of a concentration of a target substance in a sample liquid, in particular according to the method according to one of Claims 1 until 13 comprising: - a first concentrator chamber (8); - At least one liquid feed line (25) opening into the first concentrator chamber (8); and - at least one liquid discharge line (26) opening into the first concentrator chamber (8); wherein the concentrator unit (3, 33) is set up to add a first quantity of a super absorber (24) to an initial liquid volume of the sample liquid introduced into the first concentrator chamber (8) via the liquid supply line (25), or is set up to to add initial liquid volumes to the first amount of superabsorbent (4). Concentrator unit (3, 33) after claim 20 , wherein the first quantity of the superabsorbent (24) is presented in the first concentrator chamber (8). Concentrator unit (3, 33) after claim 20 , Wherein the concentrator unit (3, 33) has at least one reservoir (10) or is connected to a reservoir (10) in which the super absorber (24), in particular in the form of particles, is contained, and via a with a , in particular by means of control electronics (5), a valve-closable superabsorber supply line (23) is connected to the first concentrator chamber (8) in such a way that when the valve is open, at least part of the superabsorber (24) contained in the reservoir (10) is first quantity of the super absorber (24) enters the first concentrator chamber (8). Concentrator unit (3, 33) according to one of claims 20 until 22 , further comprising a temperature control device which is designed to control the temperature of the first concentrator chamber (8). Concentrator unit (33) according to one of claims 20 until 23 , wherein the liquid discharge line (26) can be fluidically connected to a collecting vessel (28) to discharge at least part of the liquid volume of the sample liquid present in the first concentrator chamber (8). Concentrator unit (33) according to one of claims 20 until 24 , further comprising an additional second concentrator chamber (30), wherein the liquid discharge line (26) of the first concentrator chamber (8) connects this, in particular via a collecting vessel (28), to the second concentrator chamber (30); and a further liquid discharge line (15) opening into the second concentrator chamber (30), the concentrator unit (33) being set up to deliver a second quantity of the superabsorbent (24) to a liquid volume introduced into the second concentrator chamber (8) in the to add concentrated sample liquid to the first concentrator chamber (8) or to add the liquid volume to the second quantity of superabsorbent (24). Online analysis device (1) for the detection of at least one biological target substance in a sample liquid, in particular according to the method according to one of Claims 14 until 19 , comprising: control electronics (5); a concentrator unit (3, 33) according to one of claims 20 until 25 , wherein the concentrator unit (3, 33) has a liquid outlet for concentrated sample liquid produced from the sample liquid in the concentrator unit (3, 33); and a detection unit (4) which can be fluidically connected to the liquid outlet of the concentrator unit (3, 33); and at least one transport device that can be controlled by the electronic control unit (5), e.g. a pump, which is set up to transport concentrated sample liquid from the liquid outlet of the concentrator unit (3, 33) into the detection unit (4), the electronic control unit (5) doing this is set up to use the detection unit (4) to determine the target substance in the concentrated sample liquid and/or in the sample liquid before concentration qualitatively or quantitatively based on a molecular genetic method. Online analyzer (1) after Claim 26 , further comprising a sample pump (9), wherein the electronic control system (5) is set up to control the sample pump (9) for transporting a predetermined volume of the sample liquid into the concentrator unit (8). Online analyzer (1) after Claim 26 or 27 , wherein the detection unit (4) has: - a first microfluidic unit (19) which can be fluidically connected to a liquid outlet for concentrated sample liquid of the concentrator unit and is set up to release and/or isolate nucleic acids from biomolecules contained in the concentrated sample liquid, - a second microfluidic unit (20) connected to the first microfluidic unit (19), which is set up to receive an eluate comprising the released and/or isolated nucleic acids and to amplify a target nucleic acid; and - a sensor, in particular an optical sensor, which is set up to emit a measurement signal dependent on the progress of an amplification carried out in the second microfluidic unit (20) and/or on a number of copies of the target nucleic acid in the second microfluidic unit (20). generate and output to the control electronics (5); and wherein the control electronics (5) are set up to determine the biological target substance in the liquid qualitatively and/or quantitatively on the basis of the measurement signal output by the sensor. Online analyzer (1) after claim 28 , wherein the first (19) and the second microfluidic unit (20) are arranged together in one, in particular replaceable, cartridge. Online analyzer (1) according to one of claims 28 or 29 , wherein the first microfluidic unit (19) is set up to receive the concentrated sample liquid and to mix it with one or more lysis reagents in order to release nucleic acid of the biomolecules contained in the concentrated sample liquid, then to bind the released nucleic acids to a nucleic acid-binding material, optionally under Addition of a component which enhances or mediates the binding of the nucleic acids to a nucleic acid-binding material, and washing the nucleic acids bound to the material one or more times with a washing solution. online analyzer Claim 30 , wherein the first microfluidic unit (19) is further configured to detach the nucleic acids from the nucleic acid-binding material by elution and to transport the eluate via a first microfluidic unit (19) to the second microfluidic unit (20) connecting fluid line into the second microfluidic unit (20). , and wherein the control electronics (5) are set up to control the elution and the transport of the eluate. Online analyzer (1) after claim 29 or 30 , wherein the first microfluidic unit (19) white ter is designed to receive the concentrated sample and thermally and/or chemically release the nucleic acids from the biomolecules contained in the concentrated sample in a buffer solution, wherein the control electronics (5) are set up to control the thermal treatment in the buffer solution and the To transport buffer solution with the released nucleic acids in the second microfluidic unit.

Images